Approximately half of patients infected with HIV that have access to combinational antiretroviral therapy (cART) still suffer from some form of cognitive impairment that profoundly affects quality of life. This finding conveys the need for adjunctive neuroprotective therapy to fully protect the brain from infection. Brains from HIV patients with cognitive impairment have been shown to exhibit accumulation of ceramides; one major source of ceramide is through the hydrolysis of sphingomyelin catalyzed by neutral sphingomyelinase 2 (nSmase2). Even though transient nSMase2 upregulation is part of normal brain functioning, experimental evidence is beginning to indicate that chronic nSMase2 upregulation results in negative effects including neuroinflammation and oxidative stress. While nSmase2 is emerging as an important player in the pathogenesis of HAND and other neurodegenerative diseases, the current armamentarium of nSMase2 inhibitors is inadequate to explore the role of the enzyme. Current nSMase2 inhibitors exhibit non-drug like properties including high molecular weight, poor solubility and poor pharmacokinetics. The objective of this proposal is to conduct a high throughput screen (HTS) to identify and characterize small molecule inhibitors of nSMase2. These inhibitors would be used as chemical probes to understand the role that chronic nSMase2 activation plays in HIV-associated neurocognitive disorder (HAND). Aim 1 of the project is to conduct qHTS of about 430,000 compounds from the chemical library at the NIH National Center for Advancing Translational Sciences (NCATS) with follow-up target-minus assay and orthogonal radioactivity assays to identify nSMase2 inhibitors. We will use a 1536-well fluorescence- based assay using human nSmase2 that is HTS-ready with a Z' = 0.8. Approximately 100 of the most potent and drug-like inhibitors from multiple chemotypes will be selected for evaluation in aim 2. In aim 2 we will characterize the selected inhibitors in several areas including their potential to be CNS drugs, their metabolic stability, their ability to be neuroprotective in vitro and their off target liabilities. Upon completion of am 2 we anticipate identifying 10 to 20 nSMase2 inhibitors belonging to different chemotypes with the potential to become chemical probes that will advance to aim 3. In aim 3 we will conduct preliminary SAR optimization that will focus on eliminating the most addressable liabilities encountered during inhibitor characterization in aim 2. Our criteria for a chemical probe will be: IC50 = 1 M, CNS-Multi-parameter Optimization score = 4, compound stability = 60% after 1 h incubation with mouse and human liver microsomes, dose dependent neuronal protection and = 20% inhibition of 44 targets known to be a safety risk at 10 M. Chemical probes identified through the implementation of the aims in this proposal will be available to the research community to investigate the role of nSMase2 in animal models of HAND and possibly other neurodegenerative diseases where nSMase2 is suspected to play a role.